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Der Pharma Chemica, 2013, 5(4):166-172
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ISSN 0975-413X
CODEN (USA): PCHHAX

Method development and validation of HPLC method for determination of

azithromycin
Sangita N Waghule*1, Nitin P. Jain1, Chetan J Patani2 and Aparana C. Patani3
1

Department of Pharmaceutical Chemistry, S. N. D. College of Pharmacy, Babhulgaon, Yeola, Nashik

2
Kaytross ACG Lifesciences Ltd, Ambad, Nashik
3
Department of Quality Assurance, Kaytross ACG Lifesciences Ltd, Ambad, Nashik

_____________________________________________________________________________________________
ABSTRACT
A simple, selective, precise and accurate High Performance Liquid Chromatographic Method for the analysis of
Azithromycin in its formulations was developed and validated in the present study. The mobile phase consist a
mixture of 0.0335M Phosphate Buffer (pH 7.5) and Methanol in the proportion 20:80. This was found to give sharp
peak of Azithromycin at a retention time of 8.35min. HPLC analysis of Azithromycin was carried out at a
wavelength of 210nm with a flow rate of 1.2ml/min. The linear regression analysis data for the calibration curve
showed a good linear relationship with a regression coefficient 0.997. The linear regression equation was y = 18930x
- 10493. The developed method was employed with a high degree of precision and accuracy for the analysis of
Azithromycin. The method was validated for accuracy, precision, robustness, ruggedness, specificity. The precision,
accuracy, sensitivity, short retention time and composition of the mobile phase indicated that this method is better for the
quantification of Azithromycin.
Keywords: Azithromycin, RP-HPLC Method Development, Validation.
_____________________________________________________________________________________________
INTRODUCTION
Azithromycin is a semi-synthetic macrolide antibiotic of the azalide class. Azithromycin inhibits bacterial protein
synthesis by binding to the 50S ribosomal subunit of the bacterial 70S ribosome. It inhibits peptidyltransferase
activity and interferes with amino acid translocation during the process of translation. Its effect may be bacteriostatic
or bactericidal depending on the organism and the drug concentration. Its long half life, which enables once daily
dosing and shorter administration durations, is a property distinct from other macrolides[1].
C. Barbas and L. Miguel developed a LC method for analysis of impurities in Azithromycin Tablet [3]. S.
Supattanapong and J. Konsil developed a HPLC method with electrochemical detection for analysis of
Azithromycin in Human plasma[4]. High performance liquid chromatography-electrospray ionization-tandem mass
spectrometry, LCMS/MS, Fourier-transform, Infrared transmission spectroscopy, UV, RPHPLC-UV and Reverse
Phase Ultra Performance Liquid Chromatographic techniques and amperometric electrochemical detector[2] were
developed for analysis of Azithromycin but they are expens[5-10].
The RP-HPLC method described here is simple, sensitive, and reproducible for Azithromycin determination in
Formulations with low background interference. An attempt has been made to develop and validate to ensure their
accuracy, precision and other analytical method validation parameters as mentioned in various gradients. One
method reported for the HPLC determination for developed based on the use of a C-8 column, with a suitable
mobile phase, without the use of any internal standard. For Tablet formulation the proposed method is suitable for
their analysis with virtually no interference of the usual additives presented in Tablet formulations.

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Fig. I: Structure of Azithromycin

2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy- 3,5,6,8,10,12,14-heptamethyl-15-oxo-11-{[3,4,6-trideoxy-3-(dimethylamino)-D-xylo-]oxy}-1-oxa-6-azacyclopentadec-13-yl 2,6-dideoxy-3-C-methyl-3-O-methyl--L-ribo-hexopyranoside[2]

MATERIALS AND M ETH ODS

a. Instruments:
HPLC series consisting Spectra system 2004, Spectro system P4000 pump, Autosampler AS3000, Spectro system
UV2000 detector, Thermostat column compartment connected with Win Chrome2004 software.
b. Methodology:
HPLC method is carried out by using the following conditions.
Chromatographic Conditions:
Column
: C-8, 250mm X 4.6mm, 5,
Flow rate
: 1.2 ml /min
Wavelength
: 210nm
Column temperature : 45C
Injection volume
: 20 L
Run time
: 15 minutes
Diluent
: Mobile phase
Elution
: Isocratic
Needle wash
: W a t e r : Methanol 90:10 (v/v)
c. Preparation of Mobile Phase:
The content of the Mobile Phase was prepared from filtered and degassed mixture of Phosphate buffer (4.5590gm of
Potassium dihydrogen orthophosphate in 1.0 litre Water and pH was adjusted to 7.5) and Methanol in the ratio of
20:80v/v.
d. Preparation of Azithromycin Standard Stock solution:
Weigh and transfer 50mg of Azithromycin powder into 50ml volumetric flask add 30ml of diluents and sonicate and
further filter the solution through 0.45 filter paper and make up with diluent.
e. Preparation of Sample solution:
Take 10Tablets, each containing 500mg of Azithromycin. The tablets were crushed to fine powder and amount of
powder equivalent to 100mg of Azithromycin were weighed and transferred to 100ml dried volumetric flask
dissolve the content by shaking rapidly and 100ml volumetric flask previously containing 20ml of diluents. Make
up the volume with diluent and mix well and inject immediately.
f. Procedure:
Inject 20l of blank solution, placebo solution, six times of Standard solution, Disregard peaks due to blank and
placebo.
System s u i t a b i l i t y r e q u ir e m e n t s f ro m SST solution:
Tailing factor
: NMT 2.0

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Theoretical Plates

: NLT 2000

g. Precision (Repeatability): Preparation of precision solution:

Dilute the 10ml of standard stock solution to 100ml and make-up to volume with diluent. The same procedure is
repeated to remaining six preparations. %RSD for the RT and Area are tabulated as below in Table I and II.
Table I: System Precision
S. No.
1
2
3
4
5
6
Avg.
St. dev.
%RSD

RT
8.35
8.35
8.35
8.35
8.35
8.41
8.36
0.02449
0.293

Area
123179.43
125458.40
124739.43
121780.51
123135.37
124150.46
123740.6
1314.5076
1.062

Table II: Method Precision

S. No.
1
2
3
4
5
6
Avg.
St. dev.
%RSD

RT
8.41
8.41
8.41
8.41
8.41
8.44
8.415
0.01225
0.146

Area
124209.66
122306.29
123446.69
121256.00
122487.94
122453.83
122693.4017
1018.0443
0.830

Acceptance criteria:
The %RSD of areas from six preparations precision level should not be more than 2.0%.
h. Accuracy:
The accuracy of the test method was demonstrated by preparing recovery samples (i.e. test sample with
known quantities of at the level of 115%, 125% and 150% of target concentration)
The observations of Area are tabulated as below in Table III.
Table III: Accuracy Study
Accuracy
Trial-1
Trial-2
Trial-3
Avg.
Amt. Recovered (mg)
% of Recovery

Std.
123409.55
124160.52
124646.87
124072.3133
99.97
99.97

115%Spike
141617.83
142379.43
142077.31
142024.8567
114.88
99.90

125%Spike
156607.41
156320.97
156421.72
156450.0333
124.86
99.89

150%Spike
190120.12
190108.89
190077.31
190102.1067
149.62
99.75

i. Linearity:
The Linearity of detector response for was demonstrated by prepared solutions of over the range of 100 to 800%
level of the target. Observations are tabulated in Table IV.
Table IV: Linearity Study (Preparation at 100% to 800% Level)
S. No.
1
2
3
4
5

Linear solutions (%)

100
200
400
600
800

Stock solution taken in (ml)

10
20
40
60
800

Diluted to volume (ml) with diluent

100
100
100
100
100

Area
98718.86
263076.8
458476.51
635686.46
858944.4

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Fig. II: Chromatogram of Azithromycin

Linearity of Azithromycin
1000000
y = 18930x - 10493
R = 0.997

Area

800000
600000
400000

Series1

200000

Linear (Series1)

0
10

20

40

60

80

Concentration in ppm
.
Fig. III: Linearity of Azithromycin

j. Assay:
10ml of Standard stock solution dilute to 100ml volume with diluent. Repeat the same procedure for remaining
three preparations and observations are tabulated in Table V.
Table V: Assay Study of Azithromycin
Std-1
Std-2
Std-3
Average weight
Spl-1
Spl-2
Average
LC
Standard weight
Sample weight
Standard factor
Sample factor
Standard purity
Average weight
Amount in mg
%assay

124209.66
122306.29
123446.69
123320.88
124150.46
125458.40
124804.43
500mg
50.2mg
148.8mg
0.002
0.001
98.77%
730.09mg
498.54mg
99.71%

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k. Ruggedness:
The ruggedness of test method was demonstrated by carrying out precision study in six preparations of sample on
a single batch sample by different analysts. The results of the intermediate precision study are tabulated as
below in Table VI.
Table VI: Ruggedness Study
S. No.
1
2
Avg.
Std. dev.
%RSD

RT
8.35
8.36
8.355
0.0071
0.085

Area
100093.09
100558.17
100325.63
328.8612
0.328

l. Robustness:
The robustness of test method was demonstrated by carrying out Mobile phase variation 2.0% i.e. (22:78-18:82),
flow variation 10% i.e. (1.1ml to 1.3ml/min) and Column temperature variation 5.0C i.e. (40C -50C) the
results are tabulated as below in Table VII.
Table VII: Robustness Study
S. No.
Mobile Phase-1
Mobile Phase-2
Flow rate-1
Flow rate-2
Column temp-1
Column temp-2

RT
8.54
8.35
8.54
8.30
8.33
8.35

Area
122487.94
121780.51
125087.71
124189.52
124998.84
124050.82

m. Limit of Detection (LOD):

LOD = 3 x STDEV / SLOPE
= 52.246 g/ml
n. Limit of Quantization (LOQ):
LOQ = 10 x STDEV / SLOPE
=158.321 g/ml
o. Specificity Studies:
The sample was analyzed in Specific conditions i.e. Placebo Study. Chromatogram of placebo doesnt show
interference at the retention time of Azithromycin. Therefore this method is specific for determination of
Azithromycin.
RESULTS AND DISC USS ION
The appropriate wavelength in UV region has been selected for the measurement of active ingredient in the
proposed method. This method was validated by linear fit curve and all the other parameters were calculated.
Parameters Fixation:
In developing methods, systematic study of the effects of various parameters was undertaken by varying one
parameter at a time and controlling all other parameters. The following studies were conducted for this purpose.
a) Mobile Phase Characteristics:
In order to get sharp peaks and base line separation of the components, carried out number of experiments
by varying different components like percentage of Organic phase in the mobile phase, pH of the aqueous phase,
total pH of the selected mobile phase, flow rate and column temperature by changing one at a time and keeping
all other parameters constant. The optimum conditions obtained were included in the procedure proposed.
b) Detection Characteristics:
To test whether Azithromycin has been linearly eluted from the column, different amounts of Azithromycin were
taken and analyzed by the above mentioned procedures. The peak area ratios of component areas were calculated
and the values are graphically represented in Fig. III. The linear fit of the system was illustrated graphically. Least
square regression analysis for the method was carried out for the slope, intercepts and correlation coefficient. The

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results are presented in table IV.
c) Performance Calculations:
To ascertain the system suitability for the proposed method, a number of statistical values have been calculated
with the observed readings and the results are recorded in Table VIII.
Table VIII: Performance Calculations
Parameter
Retention time (t) min
Theoretical plates (n)
Linearity Range (g/ml)
LOD (g/ml)
LOQ (g/ml)
Regression equation (y* =bc-a)
Slope (b)
Intercept (a)
Correlation coefficient
Method Precision Relative Standard Deviation (%RSD)
System Precision Relative Standard Deviation (%RSD)

RP-HPLC Method
8.35
7706.76
49.32-148.69
52.246
158.321
18930
10493
0.997
0.830
1.062

d) Method Validations:
The UV absorption maximum for Azithromycin was fixed at 210nm respectively. As the final detection was made
by the UV absorption spectrum, each method was validated by linear fit curve.
e) Precision:
The Precision of the method and system was ascertained separately from the peak area ratios obtained by
actual determination of a fixed amount of powder. The percentage of relative standard deviation is calculated for
Azithromycin and readings presented in Table I and Table II. The Precession of the assays was also determined in
terms of dilution variation in the peak areas for a set of powder solution was calculated in terms of %RSD and
the results are presented in Table V.
f) Accuracy:
To determine the accuracy of the proposed methods, different amount of samples of Azithromycin within the
linearity limits were taken and analyzed by the proposed method. The results (%RSD error) are recorded in
Table III.
g) Ruggedness:
The ruggedness of test method was demonstrated by carrying out precision study in six preparations of sample on a
single batch sample by different analysts. The results of the precision study are recorded in Table VI.
h) Robustness:
The robustness of test method was demonstrated by carrying out Mobile Phase variation 2.0% i.e. (22:78 and
18:82), flow variation 10%, i.e. (1.1ml and 1.3ml/min) and Column temperature variation 5.0C i.e. (40C50C). Results of this study are recorded in Table VII.
i) Specificity Studies:
The Specificity Studies are carried out by varying specific conditions i.e. Placebo study. Chromatogram of placebo
doesnt show interference at the retention time of Azithromycin. Therefore this method is specific for determination
of Azithromycin.
CONCLUSION
The method was found to be accurate and precise, as indicated by recovery studies close to 100 and %RSD is
not more than 2. The summary of validation parameters of proposed HPLC method is given in tables.
The simple, accurate and precise RP-HPLC method for the determination of Azithromycin as Technical and
formulation has been developed. The method may be recommended for routine and quality-control analysis
the investigated drug in formulations. The analytical solution hence, it is concluded that the analytical method is
validated and can be used for routine analysis.

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Acknowledgements
Thanks to Department of QA, SND College of Pharmacy, Babhulgaon, Yeola, Nashik and Kaytross ACG
Lifescience Ltd, Ambad, Nashik for providing laboratory facilities.
REFERENCES
th

[1] www.drugbank.ca/drugs/DB00207 (accessed on 14 June 2011).

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[5] A. Choemunng, K. Na-Bangchang, Southeast Asian J. Trop. Med. Public Health, 2010, 41, 1048-1058.
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